We propose to investigate the assembly and structure of enveloped viruses. Our proposed research will primarily examine flaviviruses. The flaviviruses comprise a genus of plus-strand RNA viruses, many of which cause significant disease in humans. Members of this family include yellow fever virus, dengue virus, and West Nile virus. We will investigate the process by which these viruses utilize their capsid protein for genome RNA recruitment, assembly, and association with the transmembrane glycoproteins. Our experience with alphavirus and flavivirus structure will support additional studies to examine the structural features of other spherical, enveloped, RNA viruses, such as rubella virus, bovine viral diarrhea virus, and coronaviruses. A multi-disciplinary approach will be employed to investigate the structure and assembly of these viruses. This approach will entail the use molecular genetics, biochemistry, and structural techniques to probe the mechanism of virus assembly and its relation to virion structure. In collaboration with the laboratories of Michael Rossmann and Tim Baker, we will use cryo-electron microscopy and X-ray crystallography to examine the structure of nucleocapsids and whole virions. We will collaborate with Carol Post to extend our structural studies on the flavivirus and pestivirus capsid proteins using NMR techniques. In parallel and independent of structural results, we will carry out biochemical studies on purified capsid proteins produced in E. coli. These studies will be combined with molecular genetic approaches including replicon technology to examine structure-function relationships in these capsid proteins. The proposed research will advance our knowledge of virus assembly, macromolecular interactions, and serve to elucidate the molecular mechanisms of virus and cellular budding. An understanding of virus assembly is expected to prove useful in the design of antiviral compounds against these various human and animal pathogens.